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Citation

Journal Of Physiology, 1992, v. 455, p. 455-469 How to Cite?

Abstract

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and pervious supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (I(SC)) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.6 ± 1.3 mV (mean ± S.E.M., n = 16), apical side negative, and a basal I(SC) Of 6.9 ± 0.9 μA cm -2 (mean ± S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 μmol 1 -1 increased the I(SC) by 3.0 ± 1.2 μA cm -2 (mean ± S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol 1 -1. Forskolin (10 μmol l -1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l -1 KCl was found to be -30 ± 14 mV (mean ± S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 μmol l -1 adrenaline by an increase in inward current at -70 mV holding potential (ΔI = -1600 ± 900 pA, mean ± S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 ± 1 mV (mean ± S.E.M., n = 16). 6. The adrenaline-induced inward current was found to be blockable by the Cl - channel blocker, DPC (0.25 mmol l -1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl -. 7. The effect of adrenaline on thec whole-cell current was found to be mimicked by forskolin and could be abolished by including GDPβS or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l -1 EGTA or 2 mmol l -1 BAPTA+ 100 μmol l -1 TMB-8 (to inhibit intracellular Ca 2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca 2+ concentration to 1 nmol l -1 or raising it to 10 μmol l -1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl - secretion in cultured human epididymal cells. The major intracellular mechanism appears to involve the classical β-adrenoceptor pathway, viz. β-adrenoceptor G(s) protein-adenylate cyclase cyclic AMP protein kinase A. However, the role of Ca 2+ in secretion in the epididymis and its interaction with cyclic AMP awaits clarification.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and pervious supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (I(SC)) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.6 ± 1.3 mV (mean ± S.E.M., n = 16), apical side negative, and a basal I(SC) Of 6.9 ± 0.9 μA cm -2 (mean ± S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 μmol 1 -1 increased the I(SC) by 3.0 ± 1.2 μA cm -2 (mean ± S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol 1 -1. Forskolin (10 μmol l -1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l -1 KCl was found to be -30 ± 14 mV (mean ± S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 μmol l -1 adrenaline by an increase in inward current at -70 mV holding potential (ΔI = -1600 ± 900 pA, mean ± S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 ± 1 mV (mean ± S.E.M., n = 16). 6. The adrenaline-induced inward current was found to be blockable by the Cl - channel blocker, DPC (0.25 mmol l -1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl -. 7. The effect of adrenaline on thec whole-cell current was found to be mimicked by forskolin and could be abolished by including GDPβS or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l -1 EGTA or 2 mmol l -1 BAPTA+ 100 μmol l -1 TMB-8 (to inhibit intracellular Ca 2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca 2+ concentration to 1 nmol l -1 or raising it to 10 μmol l -1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl - secretion in cultured human epididymal cells. The major intracellular mechanism appears to involve the classical β-adrenoceptor pathway, viz. β-adrenoceptor G(s) protein-adenylate cyclase cyclic AMP protein kinase A. However, the role of Ca 2+ in secretion in the epididymis and its interaction with cyclic AMP awaits clarification.

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Wiley-Blackwell Publishing Ltd.. The Journal's web site is located at http://www.wiley.com/bw/journal.asp?ref=0022-3751